JPWO2019088167A1 - Purification agent for sugar chain or glycopeptide and use thereof - Google Patents

Abstract

A purification agent for a glycopeptide having a sugar chain longer than a monosaccharide or a sugar chain longer than a monosaccharide, comprising a compound having a betaine structure.

Description

  The present invention relates to a sugar chain purification carrier and use thereof. More specifically, the present invention relates to a purification agent for a glycopeptide having a sugar chain longer than a monosaccharide or a sugar chain longer than a monosaccharide, a sugar chain longer than a monosaccharide, or more than a monosaccharide. A carrier for purification of a glycopeptide having a sugar chain of a length, a method for purifying a glycopeptide having a sugar chain longer than a monosaccharide or a sugar chain longer than a monosaccharide, and a sugar longer than a monosaccharide The present invention relates to a kit for purifying a glycopeptide having a sugar chain having a chain length or longer than a monosaccharide, and an apparatus for purifying a glycopeptide having a sugar chain having a length longer than a monosaccharide or a sugar chain having a length longer than a monosaccharide. . This application claims priority based on Japanese Patent Application No. 2017-210150 filed in Japan on October 31, 2017, and Japanese Patent Application No. 2017-227180 filed in Japan on November 27, 2017. The contents are incorporated herein.

  Many of the proteins that make up living organisms are modified as sugar chains and exist as glycoproteins with sugar chains bound to them. By such sugar chain modification, the protein has functions such as higher-order structure formation, intercellular signal transmission and molecular recognition, and pharmacokinetics. It is known that the structure and distribution of the sugar chain of this glycoprotein is involved in the functional expression of the protein, and the sugar chain structure changes with the onset and progression of many diseases. Structural analysis of glycoproteins plays an important role in various technical fields such as life science, medicine, drug discovery, elucidation of the pathogenesis of various diseases accompanied by sugar chain structural changes, development of disease treatment and diagnostic technologies, etc. Expected to fulfill.

  Structural analysis of glycoproteins is generally performed by analyzing glycopeptides and sugar chains obtained by decomposing glycoproteins by enzymatic digestion or the like. However, the abundance of glycopeptides and sugar chains in glycoprotein degradation products is very small, and analysis is difficult due to the peptides present in an overwhelmingly large amount. Therefore, there is a strong demand for the construction of a technique that can remove peptides to which sugar chains are not bound and specifically concentrate only the glycopeptides and sugar chains to which sugar chains are bound.

Conventionally, various techniques have been reported as a concentration technique for glycopeptides and the like. For example, concentration technology using hydrophilic interaction, concentration technology by covalent bond formation using phenylboronic acid, etc., concentration technology using interaction with lectin, titanium (eg, titanium oxide (TiO ₂ )) and zirconium In addition, a concentration technique using a chelate interaction with silver or the like has been reported (for example, see Non-Patent Document 1). Here, the concentration technique using hydrophilic interaction uses physicochemical properties such as glycopeptides, and utilizes the hydrogen bond formed between the hydroxyl group and sugar chain of Sepharose to separate Sepharose beads. To selectively capture and recover glycopeptides and the like (see, for example, Non-Patent Documents 1 and 2). Specifically, a mixture of trypsin and lysyl endopeptidase, or a digested product obtained by enzymatic digestion of a glycoprotein with chymotrypsin or the like is treated with an organic solvent (1-butanol / ethanol / water) (5: 1: 1, v / v) or the like. It is mixed with sepharose beads, glycopeptides and the like are adsorbed to the sepharose beads, washed with the above organic solvent, and then eluted with an aqueous solvent (ethanol / water) (1: 1, v / v) or the like.

Chen-Chun Chen et al., “Interaction modes and approaches to glycopeptide and glycoprotein enrichment”, Analyst, 2014, vol. 139, p.688-704 Michiko Tagiri et al., “Differential analysis of site-specific glycans on plasma and cellular fibronectins: Application of a hydrophilic affinity method for glycopeptide enrichment”, Glycobiology, 2005, vol. 15, no.12, p.1332-1340

  However, the concentration technology using Sepharose beads reported in Non-Patent Documents 1 and 2 etc. has a weak interaction between Sepharose and sugar chains, so that a glycopeptide having a small O-glucoside-linked sugar chain or a long peptide region has a low molecular weight. Since a glycopeptide having a peptide is affected by the peptide, its retention on Sepharose beads is weakened. Therefore, there is a problem that the concentration efficiency and reproducibility of glycopeptides are reduced.

  Here, for the concentration and purification of biomolecules, a column method such as a batch method or a spin column method is widely used. The batch method is a method in which a sample and a carrier are placed in the same container and stirred for a certain period of time. After the target substance is adsorbed on the carrier, the liquid phase part is removed using centrifugal force or magnetism to concentrate the target substance. is there. When Sepharose beads are applied to a batch method using a carrier, Sepharose beads absorb moisture and swell so that a clear solid-liquid separation surface is not formed. Therefore, there is a risk of sample loss such as the liquid phase part containing free peptide fragments cannot be removed effectively and the sepharose beads adsorbed with glycopeptides etc. are removed together with the liquid phase part. This is a factor in reducing the concentration efficiency and reproducibility of glycopeptides.

  In the spin column method, a sample is put into a filter cup filled with a carrier, and the sample is passed through the carrier using gravity, centrifugal force, etc., and the target substance is adsorbed, and the drainage liquid after passing through is removed. In this way, the target substance is concentrated. However, application to the spin column method as a carrier for Sepharose beads can easily achieve solid-liquid separation by using a filter cup or the like, but the surface of Sepharose beads is in a dry state. Since sepharose beads are vulnerable to drying, the recovery rate of glycopeptides is reduced, so care must be taken in handling.

  Accordingly, an object of the present invention is to provide a technique capable of specifically and efficiently capturing a hydrophilic glycopeptide and a sugar chain. It is another object of the present invention to provide a technique capable of efficiently concentrating glycopeptides and sugar chains to a high concentration from a sample contaminated with free peptide fragments by a simple operation.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors fixed the polymer in which the side chain having a betaine structure is bonded to the main chain to the insoluble support, thereby improving the hydrophilicity of the carrier. Has been found to be effective. Furthermore, the carrier has a property of strongly retaining a glycopeptide and a sugar chain having high hydrophilicity by hydrophilic interaction, and by utilizing such a property, the glycopeptide and the sugar chain can be efficiently and easily operated. It was found that it can be concentrated to high purity and high concentration.

  That is, the present invention includes a carrier, a glycopeptide concentration method using the carrier, a sugar chain concentration method, a kit for glycopeptide concentration, a kit for sugar chain concentration, a device for glycopeptide concentration, and a sugar chain An apparatus for concentration is provided, which includes the following configuration in detail.

［式（１）中、Ｚは、２級アミノ基、３級アミノ基、４級アンモニウム基及びイミノ基からなる群より選択されるカチオン性基を表し、Ｌは炭素数１〜１０のアルキレン基を表し、Ａは、リン酸基、カルボキシル基、ホスホン酸基、ホスフィン酸基、スルホン酸基、スルフィン基、スルフェン基、水酸基、チオール基及びボロン酸基からなる群より選択されるアニオン性基を表す。］
〔３〕前記カチオン性基が４級アンモニウム基である、〔１〕又は〔２〕に記載の精製剤。
〔４〕前記アニオン性基がリン酸基である、〔１〕〜〔３〕のいずれかに記載の精製剤。
〔５〕前記ベタイン構造が、ホスホリルコリン基である、〔１〕〜〔４〕のいずれかに記載の精製剤。
〔６〕前記化合物が、ベタイン構造を有する側鎖が主鎖に結合したポリマーである、〔１〕〜〔５〕のいずれか一項に記載の精製剤。
〔７〕前記ポリマーが、（メタ）アクリル化合物を含む単量体の重合体である、〔６〕に記載の精製剤。
〔８〕〔１〕〜〔５〕のいずれかに記載の精製剤が、不溶性支持体に固定された、単糖以上の長さの糖鎖又は単糖以上の長さの糖鎖を有する糖ペプチドの精製用担体。
〔９〕〔６〕又は〔７〕に記載の精製剤が、不溶性支持体に固定された、単糖以上の長さの糖鎖又は単糖以上の長さの糖鎖を有する糖ペプチドの精製用担体。
〔１０〕前記不溶性支持体に固定された前記ポリマーの重量が、前記不溶性支持体の単位表面積（ｍ^２）当たり０．５ｍｇ〜１．５ｍｇである、〔９〕に記載の精製用担体。
〔１１〕前記不溶性支持体が、無機物質により構成されている、〔８〕〜〔１０〕のいずれかに記載の精製用担体。
〔１２〕比重が、１．０５〜３．００である、〔８〕〜〔１１〕のいずれかに記載の精製用担体。
〔１３〕形状が球状であり、平均粒径が０．５μｍ〜１００μｍである、〔８〕〜〔１２〕のいずれかに記載の精製用担体。
〔１４〕〔１〕〜〔７〕のいずれかに記載の精製剤又は〔８〕〜〔１３〕のいずれかに記載の精製用担体と、単糖以上の長さの糖鎖又は単糖以上の長さの糖鎖を有する糖ペプチドと有機溶剤を含む試料とを接触させ、前記精製剤又は精製用担体に前記糖鎖又は前記糖ペプチドを吸着させることと、上記精製剤又は精製用担体を水に接触させ、単糖以上の長さの糖鎖又は単糖以上の長さの糖鎖を有する糖ペプチドを溶出させることと、を含む、単糖以上の長さの糖鎖又は単糖以上の長さの糖鎖を有する糖ペプチドの精製方法。
〔１５〕〔１〕〜〔７〕のいずれかに記載の精製剤又は〔８〕〜〔１３〕のいずれかに記載の精製用担体、及び、キット使用のためのプロトコル情報を含む、単糖以上の長さの糖鎖又は単糖以上の長さの糖鎖を有する糖ペプチド精製のためのキット。
〔１６〕〔１〕〜〔７〕のいずれかに記載の精製剤又は〔８〕〜〔１３〕のいずれかに記載の精製用担体を収容した容器を保持する容器保持部と、前記容器に試薬類を導入する試薬導入部と、を備える、単糖以上の長さの糖鎖又は単糖以上の長さの糖鎖を有する糖ペプチド精製のための装置。[1] A purification agent for a glycopeptide having a sugar chain longer than a monosaccharide or a sugar chain longer than a monosaccharide, comprising a compound having a betaine structure.
[2] The purifying agent according to [1], wherein the betaine structure is a structure represented by the following formula (1).

[In the formula (1), Z represents a cationic group selected from the group consisting of a secondary amino group, a tertiary amino group, a quaternary ammonium group and an imino group, and L represents an alkylene group having 1 to 10 carbon atoms. A represents an anionic group selected from the group consisting of a phosphate group, a carboxyl group, a phosphonic acid group, a phosphinic acid group, a sulfonic acid group, a sulfinic group, a sulfenic group, a hydroxyl group, a thiol group and a boronic acid group. Represent. ]
[3] The purifying agent according to [1] or [2], wherein the cationic group is a quaternary ammonium group.
[4] The purification agent according to any one of [1] to [3], wherein the anionic group is a phosphate group.
[5] The purifying agent according to any one of [1] to [4], wherein the betaine structure is a phosphorylcholine group.
[6] The purification agent according to any one of [1] to [5], wherein the compound is a polymer in which a side chain having a betaine structure is bonded to a main chain.
[7] The purifying agent according to [6], wherein the polymer is a polymer of a monomer containing a (meth) acrylic compound.
[8] A saccharide having a sugar chain having a length longer than a monosaccharide or a sugar chain having a length longer than a monosaccharide, wherein the purification agent according to any one of [1] to [5] is fixed to an insoluble support. A carrier for peptide purification.
[9] Purification of a glycopeptide having a sugar chain longer than a monosaccharide or a sugar chain longer than a monosaccharide, wherein the purification agent according to [6] or [7] is fixed to an insoluble support Carrier.
[10] The purification carrier according to [9], wherein the weight of the polymer fixed to the insoluble support is 0.5 mg to 1.5 mg per unit surface area (m ² ) of the insoluble support.
[11] The purification carrier according to any one of [8] to [10], wherein the insoluble support is composed of an inorganic substance.
[12] The purification carrier according to any one of [8] to [11], wherein the specific gravity is 1.05 to 3.00.
[13] The purification carrier according to any one of [8] to [12], which has a spherical shape and an average particle diameter of 0.5 μm to 100 μm.
[14] The purification agent according to any one of [1] to [7] or the purification carrier according to any one of [8] to [13], and a sugar chain having a length longer than a monosaccharide or more than a monosaccharide A sugar peptide having a sugar chain of a length of length and a sample containing an organic solvent are brought into contact with each other to adsorb the sugar chain or the glycopeptide to the purification agent or purification carrier, and the purification agent or purification carrier Contacting with water and eluting a glycopeptide having a sugar chain longer than a monosaccharide or a sugar chain longer than a monosaccharide, and having a sugar chain longer than a monosaccharide or more than a monosaccharide A method for purifying a glycopeptide having a sugar chain of a length of
[15] A monosaccharide comprising the purification agent according to any one of [1] to [7] or the purification carrier according to any one of [8] to [13] and protocol information for using the kit A kit for purifying a glycopeptide having a sugar chain having the above length or a sugar chain having a length longer than a monosaccharide.
[16] A container holding unit for holding a container containing the purification agent according to any one of [1] to [7] or the purification carrier according to any one of [8] to [13]; An apparatus for purifying a glycopeptide having a sugar chain longer than a monosaccharide or a sugar chain longer than a monosaccharide, comprising a reagent introduction part for introducing reagents.

[P1] A carrier in which a polymer in which a side chain having a betaine structure is bonded to a main chain is fixed to an insoluble support.

  According to the said structure, the support | carrier with which hydrophilicity was raised effectively can be provided by fixing the polymer which the side chain which has a betaine structure couple | bonds with the principal chain to an insoluble support body. The carrier has a characteristic that it can strongly retain a highly hydrophilic glycopeptide or sugar chain by hydrophilic interaction. Moreover, the carrier can capture a glycopeptide having an O-glycoside-linked sugar chain or a glycopeptide having a long peptide region without being affected by the peptide. Therefore, the carrier can concentrate a glycopeptide and a sugar chain efficiently, with high purity and at a high concentration by a simple operation by utilizing such properties, and a carrier having excellent operability and functionality. Can be provided.

[P2] The carrier of [P1] above, wherein the betaine cation moiety contains a quaternary ammonium group.
[P3] The carrier of [P1] or [P2] above, wherein the betaine anion moiety contains a phosphate group.
[P4] The carrier according to any one of [P1] to [P3], wherein the betaine structure is phosphorylcholine.
[P5] The carrier according to any one of [P1] to [P4], wherein the polymer is composed of a polymer derived from a monomer containing a (meth) acrylic compound.

  According to the above configuration, it is possible to provide a carrier using a polymer that has very high hydrophilicity and biocompatibility, and is easy to obtain and synthesize. A carrier that can be concentrated to a high purity and a high concentration can be provided.

[P6] Any of [P1] to [P5] above, wherein the weight of the polymer bonded to the insoluble support is 0.5 mg to 1.5 mg per unit surface area (m ² ) of the insoluble support. Carrier.
[P7] The carrier according to any one of [P1] to [P6], wherein the insoluble support is composed of an inorganic substance.
[P8] The carrier according to any one of [P1] to [P7], wherein the specific gravity is 1.05 to 3.00.
[P9] The carrier according to any one of [P1] to [P8] above, wherein the shape is spherical and the average particle diameter is 0.5 μm to 100 μm.

  According to the above configuration, it is possible to provide a carrier having good sedimentation properties and dispersibility, and more excellent operability. In addition, when applied for the concentration of glycopeptides and sugar chains, a carrier that is more excellent in terms of the efficiency of capturing the glycopeptides and sugar chains and the separation efficiency from free peptide fragments and the like can be provided.

[P10] The carrier according to any one of [P1] to [P9] for concentration of glycopeptide.

  According to the above configuration, a carrier for concentrating glycopeptides can be provided, and the carrier has a characteristic of strongly retaining glycopeptides with high hydrophilicity by hydrophilic interaction. Efficient, high purity and high concentration.

[P11] The carrier according to any one of [P1] to [P9] for concentration of sugar chains.

  According to the above configuration, a carrier for concentrating sugar chains can be provided, and the carrier has a characteristic of strongly holding a sugar chain having a high degree of hydrophilicity by hydrophilic interaction. Efficient, high purity and high concentration.

[P12] A method for concentrating glycopeptides, the method comprising concentrating glycopeptides using the carrier according to any one of [P1] to [P10].
[P13] The method for concentrating glycopeptides according to [P12] above, wherein the step of concentrating the glycopeptide is a concentration step by a batch method or a spin column method.

  According to the said structure, the concentration method of the glycopeptide which uses the support | carrier of this invention can be provided, and glycopeptide can be efficiently concentrated with high purity and high concentration by simple operation. In addition, the glycopeptide can be suitably applied to a batch method, a spin column method, and the like, and the glycopeptide can be concentrated efficiently, with high purity and at a high concentration by a simpler operation.

[P14] A method for concentrating sugar chains, the method comprising concentrating sugar chains using the carrier according to any one of [P1] to [P9] and [P11].
[P15] The method for concentrating sugar chains according to the above [P14], wherein the step of concentrating the sugar chains is a concentration step by a batch method or a spin column method.

  According to the said structure, the concentration method of the sugar chain using the support | carrier of this invention can be provided, and a sugar chain can be efficiently concentrated with high purity and high concentration by simple operation. Further, it can be suitably applied to a batch method, a spin column method, and the like, and sugar chains can be concentrated efficiently, with high purity and at a high concentration by a simpler operation.

[P16] A kit for concentrating glycopeptides comprising the carrier of any of the above [P1] to [P10] and protocol information for using the kit.

  According to the above configuration, the glycopeptide can be concentrated more easily by using a carrier and information necessary for the concentration of the glycopeptide as a kit.

[P17] A kit for concentrating sugar chains comprising the carrier of any of the above [P1] to [P9] and [P11] and protocol information for using the kit.

  According to the said structure, a sugar chain can be concentrated more simply by making the carrier and information required for sugar chain concentration into a kit.

[P18] A container holding part for holding a container in which the carrier of any of the above [P1] to [P10] is introduced and which can contain a sample containing a glycopeptide, and reagent introduction for introducing reagents into the container And an apparatus for concentrating glycopeptides.
[P19] The apparatus for concentrating glycopeptides of [P18], further comprising a solid-liquid separation unit for solid-liquid separation of the contents in the container.

  According to the said structure, concentration of a glycopeptide can be performed more simply by apparatus-izing the support | carrier and members required for concentration of a glycopeptide.

[P20] A container holding part for holding a container in which a carrier of any of the above [P1] to [P9] and [P11] is introduced and can contain a sample containing a sugar chain, and reagents in the container An apparatus for concentrating sugar chains, comprising a reagent introduction part to be introduced.
[P21] The apparatus for concentrating sugar chains according to [P20], further comprising a solid-liquid separation unit for solid-liquid separation of the contents in the container.

  According to the above configuration, sugar chains can be concentrated more easily by incorporating the carriers and members necessary for sugar chain concentration.

  According to the present invention, it is possible to provide a technique capable of specifically and efficiently capturing a hydrophilic glycopeptide and a sugar chain.

It is the figure which represented typically an example of the support | carrier which concerns on this embodiment. It is the figure which represented typically an example of the synthesis | combining method of the polymer layer of the support | carrier which concerns on this embodiment. It is a chromatogram which shows the result of Example 2 which examined concentration of the glycopeptide using the support | carrier which concerns on this embodiment. It is a mass spectrum (MS) chart which shows the result of Example 2 which examined concentration of the glycopeptide using the support | carrier which concerns on this embodiment. It is a graph which shows the result of Example 3 which examined concentration of the sugar_chain | carbohydrate using the support | carrier which concerns on this embodiment, a vertical axis | shaft shows a peak area and a horizontal axis shows glucose number. It is a graph which shows the result of the comparative example 2 which examined concentration of the sugar chain using a cleanup column, a vertical axis | shaft shows a peak area and a horizontal axis shows the glucose number. It is a graph which shows the result of the comparative example 3 which examined concentration of the sugar_chain | carbohydrate using graphite carbon, a vertical axis | shaft shows a peak area and a horizontal axis shows glucose number. It is a graph which shows the result of Example 4 which examined concentration of the sugar_chain | carbohydrate using the support | carrier which concerns on this embodiment, a vertical axis | shaft shows a peak area and a horizontal axis shows glucose number. It is a schematic diagram explaining the refinement | purification apparatus of the sugar_chain | carbohydrate or glycopeptide of this embodiment.

  Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described below.

(Purifying agent)
In one embodiment, the present invention provides a purification agent for a glycopeptide having a sugar chain longer than a monosaccharide or a sugar chain longer than a monosaccharide, comprising a compound having a betaine structure. As will be described later in Examples, the purification agent of the present embodiment can efficiently purify a glycopeptide having a sugar chain having a length longer than a monosaccharide or a sugar chain having a length longer than a monosaccharide.

  The purification agent of this embodiment may be called a purification agent for a glycopeptide having a sugar chain longer than a monosaccharide or having a sugar chain longer than a monosaccharide, containing a compound having a betaine structure as an active ingredient. it can. That is, as long as the purification agent of the present embodiment includes a compound having a betaine structure, a sugar chain or a monosaccharide having a length longer than that of a monosaccharide, even in a mixture with a compound having no betaine structure. A glycopeptide having a sugar chain of the length can be purified.

  The purification agent of this embodiment may be immobilized on an insoluble support to form a carrier for purifying a glycopeptide having a sugar chain longer than a monosaccharide or a sugar chain longer than a monosaccharide. . In the present specification, the “purification carrier” is sometimes simply referred to as “carrier”.

  The above-mentioned betaine structure may be a structure represented by the following formula (1).

［式（１）中、Ｚは、２級アミノ基、３級アミノ基、４級アンモニウム基及びイミノ基からなる群より選択されるカチオン性基を表し、Ｌは炭素数１〜１０のアルキレン基を表し、Ａは、リン酸基、カルボキシル基、ホスホン酸基、ホスフィン酸基、スルホン酸基、スルフィン基、スルフェン基、水酸基、チオール基及びボロン酸基からなる群より選択されるアニオン性基を表す。］

[In the formula (1), Z represents a cationic group selected from the group consisting of a secondary amino group, a tertiary amino group, a quaternary ammonium group and an imino group, and L represents an alkylene group having 1 to 10 carbon atoms. A represents an anionic group selected from the group consisting of a phosphate group, a carboxyl group, a phosphonic acid group, a phosphinic acid group, a sulfonic acid group, a sulfinic group, a sulfenic group, a hydroxyl group, a thiol group and a boronic acid group. Represent. ]

  The compound having the betaine structure described above may be betaine or a polymer in which a side chain having a betaine structure is bonded to the main chain.

  As will be described later in Examples, the purifying agent of this embodiment has a remarkably high performance for purifying short sugar chains as compared to conventional purifying agents. “Purification” can also be called “concentration”.

  In the present specification, the main chain refers to the longest carbon chain in the polymer structure, and a structure branched from the main chain is referred to as a side chain. Moreover, in this specification, a sugar chain shall contain a monosaccharide. Therefore, a sugar chain having a length longer than a monosaccharide includes a sugar chain having a length longer than a monosaccharide, a disaccharide, a trisaccharide, and a tetrasaccharide. The molecular weight of a sugar chain having a length longer than a monosaccharide may be, for example, about 150 to 3000.

  In the carrier according to the embodiment, a polymer is immobilized on an insoluble support, and the polymer has a side chain having a betaine structure bonded to a main chain. The carrier according to the embodiment has a betaine structure, so that the hydrophilicity is extremely increased, and a glycopeptide or sugar chain having a high hydrophilicity can be strongly retained by a hydrophilic interaction.

  In the carrier according to the embodiment, a polymer is immobilized on an insoluble support, and preferably, the polymer covers all or a part of the surface of the insoluble support to form a polymer layer. Here, the coating means that the polymer adheres to the surface of the insoluble support. FIG. 1 schematically shows an example of the carrier according to the embodiment. In the carrier of FIG. 1, a polymer layer having a betaine structure is formed on the surface of an insoluble support.

  The polymer layer may contain a polymer not having a betaine structure in addition to a polymer having a side chain having a betaine structure bonded to the main chain. As long as the betaine structure exists on the surface of the carrier according to the embodiment, a glycopeptide having a sugar chain having a length longer than a monosaccharide or a sugar chain having a length longer than a monosaccharide can be purified.

  The insoluble support is a substrate insoluble in water and an organic solvent used in the purification process of the sugar chain or glycopeptide, and is not particularly limited as long as the polymer according to the embodiment can be immobilized. Can be used. The material of the insoluble support may be either an inorganic substance or an organic substance, or may be a composite substance using them together. Inorganic substances include silicon compounds such as silica, glass such as silicate glass, iron oxide (ferrite, magnetite, etc.), oxides such as alumina, titania, zirconia, iron, copper, gold, silver, platinum, cobalt, Examples thereof include metals such as aluminum, palladium, iridium, and rhodium and alloys thereof, and carbon materials such as graphite. These may be used individually by 1 type and may be used in combination of 2 or more type. Examples of the organic substance include synthetic polymers such as crosslinked polyvinyl alcohol, crosslinked polyacrylate, crosslinked polyacrylamide, and crosslinked polystyrene, polysaccharides such as crosslinked sepharose, crystalline cellulose, crosslinked cellulose, crosslinked amylose, crosslinked agarose, and crosslinked dextran. It is done. These may be used individually by 1 type and may be used in combination of 2 or more type. Moreover, the polymer itself in which the side chain having a betaine structure is bonded to the main chain may form an insoluble support.

  As the insoluble support, it is preferable to use an inorganic substance, and particularly preferably a silicon compound, especially silica. In general, an organic substance that can be an insoluble support has a specific gravity of about 1, and the difference in specific gravity from a glycopeptide solution or a sugar chain solution is small, so that solid-liquid separation is often complicated. By using an inorganic substance, for example, when the carrier according to the embodiment is applied for concentration of glycopeptides or sugar chains, the solid-liquid separation can be easily and easily performed, and the glycopeptide or sugar chain captured by the carrier is used. And impurities such as free peptide fragments can be effectively separated. Thereby, it can contribute to the improvement of concentration efficiency. In addition, the inorganic substance can impart an appropriate strength to the carrier.

  The insoluble support may have voids such as a porous body and a hollow body. For example, examples of the porous body include monolith type silica. Monolith type silica has a micrometer-size three-dimensional network pore (macropore) and a silica skeleton that forms a three-dimensional network structure with a nanometer-size pore (mesopore). It is a porous structure. The pore diameter of the micropores can be independently controlled, for example, in a range of 1 μm to 100 μm, preferably 1 μm to 50 μm, and the mesopore diameter is, for example, 1 nm to 100 nm, preferably 1 nm to 70 nm. By using the insoluble support having such voids, the carrier according to the embodiment has a large specific surface area, and the amount of the purification agent that can be immobilized on the surface of the insoluble support can be increased. As a result, when the carrier of the embodiment is used for concentrating glycopeptides and sugar chains, the contact efficiency with the glycopeptides and sugar chains can be improved, and the glycopeptides and sugar chains can be efficiently captured. It can contribute to improvement. It can also be used to adjust the specific gravity of the insoluble support described later.

  When the purification agent is a polymer, the polymer may be a polymer of a polymerizable monomer. The polymerizable monomer is not particularly limited as long as it is a monomer capable of forming a polymer by a polymerization reaction, and is preferably a (meth) acryl compound having a (meth) acryloyl group, for example, (meth) Acrylic acid esters and derivatives thereof are included. Furthermore, a compound having a vinyl group, an allyl group, an α-alkoxymethylacryloyl group, a maleic acid residue, a fumaric acid residue, an itaconic acid residue, a crotonic acid residue, an isocrotonic acid residue, a citraconic acid residue, and the like Examples thereof include, but are not limited to, derivatives thereof. A polymerizable monomer may be used individually by 1 type, and may use 2 or more types together. In addition, “(meth) acryloyl group” represents “acryloyl group” or “methacryloyl group”, and “(meth) acryl” represents “acryl” or “methacryl”.

  The side chain of the polymer immobilized on the insoluble support according to the embodiment is a molecular chain branched from the main chain composed of the polymer of the polymerizable monomer described above, and part or all of which has a betaine structure. Have. The betaine structure means a structure having a cation moiety and an anion moiety at separate and non-adjacent positions in the same molecule.

The cationic site is a positively charged atomic group and means a so-called cationic group. Examples of the cationic group include a primary amino group, a secondary amino group (—NHR), a tertiary amino group (—NR ₂ ), a quaternary ammonium group (—NR ₃ ⁺ ), and an imino group. However, it is not limited to these. R in the secondary amino group, tertiary amino group, and quaternary ammonium group is an alkyl group or an aryl group, and when one group has a plurality of Rs, they may be the same or different, for example, , Methyl group, ethyl group, propylene group and the like, but are not limited thereto. A quaternary ammonium group is preferred, and a trimethylammonium group is particularly preferred. In addition, a salt form in which a salt is formed with fluoride ion, chloride ion, bromide ion, iodide ion, hydrochloric acid ion, acetate ion, sulfate ion, hydrofluoric acid ion, carbonate ion or the like is also a cationic group. include.

  An anion site is a negatively charged atomic group and means a so-called anionic group. Examples of the anionic group include a phosphoric acid group, a phosphonic acid group, a phosphinic acid group, a sulfonic acid group, a sulfinic group, a sulfenic group, a carboxyl group, a hydroxyl group, a thiol group, and a boronic acid group. It is not limited to. Preferably, it is a phosphate group. Moreover, the anionic group includes salts in the form of salts with alkali metal ions such as sodium ions and potassium ions and alkaline earth metal ions such as calcium ions.

  The betaine structure is not particularly limited as long as it has the above-described cation moiety and anion moiety, and the combination of the cation moiety and the anion moiety is not particularly limited. Preferably, the cation moiety is a quaternary ammonium group and the anion moiety is a phosphate group.

  The polymer immobilized on the insoluble support according to the embodiment is not particularly limited as long as the side chain having a betaine structure is bonded to the main chain composed of a polymer of a polymerizable monomer. Accordingly, the polymer may be a copolymer of a polymerizable monomer having a cationic site and a polymerizable monomer having an anionic site, in addition to a homopolymer of a polymerizable monomer having a betaine structure. Further, it may be a copolymer including a polymerizable monomer having no charge, and the solubility of the polymer in water can be controlled by including such a polymerizable monomer. The copolymer means a polymer obtained from two or more types of monomers, and may be any of an alternating copolymer, a block copolymer, a random copolymer, a graft copolymer, and the like. . Therefore, the betaine structure may be introduced for every monomer unit of the polymer, may be introduced for every fixed unit of the monomer unit, or may be introduced at random.

  Preferably, the polymer side chain is a homopolymer of a polymerizable monomer having a betaine structure. In this case, the polymerizable monomer having a betaine structure has an anion portion and a cation portion in the same molecular chain. The linker that connects the two is not particularly limited as long as it has a divalent or higher valent group, and a known linker can be used. Preferably, it is an alkylene linker, and examples of the alkylene linker include alkylene linkers having 1 to 10 carbon atoms, preferably 2 to 5 carbon atoms.

  Examples of such a polymerizable monomer having a betaine structure include a phosphobetaine monomer having a phosphobetaine group such as a phosphorylcholine group, a carboxybetaine monomer having a carboxybetaine group, and a sulfobetaine group. Examples include sulfobetaine monomers, but are not limited thereto. Preferably, it is a phosphobetaine monomer, and among them, a phosphobetaine monomer having a phosphorylcholine group.

  As the phosphobetaine monomer, a polymerizable monomer having a phosphorylcholine group is preferable. For example, 2- (meth) acryloyloxyethylphosphorylcholine, 2- (meth) acryloyloxyethoxyethylphosphorylcholine, 6- (meth) Examples include acryloyloxyhexyl phosphorylcholine, 10- (meth) acryloyloxyethoxynonyl phosphorylcholine, 2- (meth) acryloyloxypropyl phosphorylcholine, 2- (meth) acryloyloxybutyl phosphorylcholine, and the like. Among these, 2- (meth) acryloyloxyethyl phosphorylcholine is particularly preferable from the viewpoint of availability. Furthermore, as phosphobetaine monomers, for example, dimethyl (2-methacryloyloxyethyl) (2-phosphonatoethyl) aminium, dimethyl (2-acryloyloxyethyl) (2-phosphonatoethyl) aminium, dimethyl (2-methacryloyloxyethyl) (3-phosphonatopropyl) aminium, dimethyl (2-acryloyloxyethyl) (3-phosphonatopropyl) aminium, dimethyl (2-methacryloyloxyethyl) (4-phosphonatobutyl) aminium, dimethyl (2-acryloyloxyethyl) ( 4-phosphonatobutyl) aminium, dimethyl (2-methacryloyloxyethyl) (phosphonatomethyl) aminium, dimethyl (2-acryloyloxyethyl) (phosphonatomethyl) aminium .

  Examples of carboxybetaine monomers include dimethyl (2-methacryloyloxyethyl) (2-carboxylatoethyl) aminium, dimethyl (2-acryloyloxyethyl) (2-carboxylatoethyl) aminium, dimethyl (2-methacryloyl). Oxyethyl) (3-carboxylatopropyl) aminium, dimethyl (2-acryloyloxyethyl) (3-carboxylatopropyl) aminium, dimethyl (2-methacryloyloxyethyl) (4-carboxylatobutyl) aminium, dimethyl (2- Acryloyloxyethyl) (4-carboxylatobutyl) aminium, dimethyl (2-methacryloyloxyethyl) (carboxylatomethyl) aminium, dimethyl (2-acryloyloxyethyl) (carboxyla) Methyl) aminium the like.

  Examples of the sulfobetaine monomer include dimethyl (2-methacryloyloxyethyl) (2-sulfonatoethyl) aminium, dimethyl (2-acryloyloxyethyl) (2-sulfonatoethyl) aminium, and dimethyl (2-methacryloyl). Oxyethyl) (3-sulfonatopropyl) aminium, dimethyl (2-acryloyloxyethyl) (3-sulfonatopropyl) aminium, dimethyl (2-methacryloyloxyethyl) (4-sulfonatobutyl) aminium, dimethyl (2-acryloyloxy) And ethyl) (4-sulfonatobutyl) aminium, dimethyl (2-methacryloyloxyethyl) (sulfonatomethyl) aminium, dimethyl (2-acryloyloxyethyl) (sulfonatomethyl) aminium, and the like.

The weight of the polymer bonded to the insoluble support is preferably about 0.5 mg to 1.5 mg, particularly 0.6 mg to 1.3 mg, per unit surface area (m ² ) of the insoluble support. In the following, it is further preferably 0.7 mg or more and 1.2 mg or less. When the polymer weight per unit surface area is in the above range, handling during polymer synthesis is good, good contact efficiency with glycopeptides and sugar chains can be secured, and glycopeptides and sugar chains are efficiently captured. be able to.

  The carrier according to the embodiment preferably has a specific gravity of about 1.05 to 3.00, particularly 1.1 to 2.7, and more preferably 1.5 to 2.5. When the specific gravity is less than the lower limit value, the sedimentation property is lowered, and when the specific gravity is more than the upper limit value, the dispersibility is deteriorated. Therefore, when the specific gravity of the carrier according to the embodiment is within the above range, when applied for the concentration of glycopeptides or sugar chains, etc. Solid-liquid separation can be performed easily and simply, and the glycopeptide and sugar chain captured by the carrier can be effectively separated from contaminants such as free peptide fragments. Moreover, since the dispersibility is good, the contact efficiency with the glycopeptide or sugar chain is improved, and the glycopeptide or sugar chain can be efficiently captured. Therefore, it is possible to provide a carrier that is excellent in terms of operability, and in the case of application for the concentration of glycopeptides and sugar chains, the separation from free peptide fragments and the like and the capture efficiency of glycopeptides and sugar chains. However, an excellent carrier can be provided.

  The shape of the carrier in the embodiment is not particularly limited, and may be any known shape. Examples thereof include spherical shapes such as beads, plate shapes such as substrates and multiwell plates, sheets such as sheets and films, membranes such as membranes, and fibers. The carrier can also be called a solid phase. The shape is preferably a spherical shape or the like that can be easily handled. In the case of a spherical shape, the average particle size is preferably about 0.5 μm to 100 μm, and particularly preferably 1 μm to 50 μm, or 1 μm to 10 μm. Particularly preferably, it is 3 μm or more and 10 μm or less. If the average particle size is less than the lower limit, it becomes difficult to collect the carrier by centrifugation or filtration, and when the carrier is packed in a column or the like, the liquid permeability becomes poor and it is necessary to apply a large pressure during the liquid passage. Occurs. On the other hand, when the average particle size exceeds the upper limit, the contact area between the carrier and the sample solution decreases, the capture efficiency of glycopeptides and sugar chains decreases, and the concentration efficiency decreases. Therefore, when the average particle size of the carrier according to the embodiment is within the above range, it is possible to provide a carrier that is excellent in terms of operability, and when it is applied for concentration of glycopeptides and sugar chains, it is free. An excellent carrier can be provided in terms of separation from peptide fragments and the like and capture efficiency of glycopeptides and sugar chains. The average particle diameter can be measured by, for example, a particle size distribution meter.

  The carrier according to the embodiment may be used in a state of being filled in a container such as a filter cup such as a spin column, each well of a multiwell plate, each well of a filter plate, or a microtube.

  The polymer can be obtained by polymerizing the polymerizable monomer, but the polymer polymerization method is not particularly limited and can be appropriately selected according to the type of the polymerizable monomer. Preferably, it is radical polymerization.

  The immobilization of the polymer on the insoluble support may be performed using either physical adsorption or chemical bonding. Preferably, it is a chemical bond from the viewpoint of stability, and elution of the polymer from the insoluble support can be suppressed. Further, the polymer may be immobilized on the surface of the insoluble support by polymerizing a polymerizable monomer on the surface of the insoluble support, or the polymer that has been polymerized in advance may be immobilized on the surface of the insoluble support.

  When immobilizing a polymer on the surface of an insoluble support by polymerizing a polymerizable monomer on the surface of the insoluble support, for example, an insolubility in which a polymerization start point is introduced on the surface of the insoluble support and a polymerization start point is introduced The polymer can be grown from the polymerization starting point by immersing the support in the polymerizable monomer solution and adding a polymerization initiator. Thereby, the polymer can be immobilized on the surface of the insoluble support by chemical bonding. As a polymerization starting point, a polymerizable functional group, a chain transfer group, a dormant species in living radical polymerization, or the like can be used.

  Examples of the polymerizable functional group include a vinyl group, an allyl group (2-propenyl group), a (meth) acryloyl group, an epoxy group, and a styrene group. Examples of the chain transfer group include a mercapto group and an amino group, and a mercapto group is preferable in terms of excellent reactivity.

  The method for introducing the polymerizable functional group or chain transfer group onto the surface of the insoluble support is not particularly limited, but it is preferable to use a silane coupling agent having a polymerizable functional group or chain transfer group.

  Examples of the silane coupling agent having a polymerizable functional group include (3-methacryloxypropyl) dimethylmethoxysilane, (3-methacryloxypropyl) diethylmethoxysilane, (3-methacryloxypropyl) dimethylethoxysilane, (3 -Methacryloxypropyl) diethylethoxysilane, (3-methacryloxypropyl) methyldimethoxysilane, (3-methacryloxypropyl) ethyldimethoxysilane, (3-methacryloxypropyl) methyldiethoxysilane, (3-methacryloxypropyl) Ethyldiethoxysilane, (3-methacryloxypropyl) trimethoxysilane, (3-methacryloxypropyl) triethoxysilane, and the like can be mentioned. From the viewpoint of reactivity and availability, (3-methacryloxypropyl) tri Tokishishiran or (3-methacryloxypropyl) triethoxysilane are preferred. These silane coupling agents can be used alone or in combination of two or more.

  Examples of the silane coupling agent having a chain transfer group include (3-mercaptopropyl) trimethoxysilane, (3-mercaptopropyl) methyldimethoxysilane, (3-mercaptopropyl) dimethylmethoxysilane, and (3-mercaptopropyl) tri. Ethoxysilane, (3-mercaptopropyl) methyldiethoxysilane, (3-mercaptopropyl) dimethylethoxysilane, (mercaptomethyl) trimethoxysilane, (mercaptomethyl) methyldimethoxysilane, (mercaptomethyl) dimethylmethoxysilane, (mercapto) Methyl) triethoxysilane, (mercaptomethyl) methyldiethoxysilane, (mercaptomethyl) dimethylethoxysilane, and the like. From the availability, (3-mercaptopropyl) trimethoxysilane and (3-Mercaptopropyl) triethoxysilane is preferred. These silane coupling agents can be used alone or in combination of two or more.

  Introduction of a polymerizable functional group or chain transfer group into an insoluble support using a silane coupling agent having a polymerizable functional group or chain transfer group is, for example, a silane coupling agent and an insoluble support. This can be done by forming a covalent bond with the functional group on the surface. For example, when alkoxysilanes such as trimethoxysilanes and triethoxysilanes are used as silane coupling agents, silanol groups generated by hydrolysis are hydroxyl groups, amino groups, carbonyl groups, silanols on the surface of the insoluble support. It can be carried out by dehydration condensation with a group or the like to form a covalent bond.

  After introducing a polymerizable functional group or chain transfer group to the surface of the insoluble support, a polymer layer is formed on the surface of the insoluble support by mixing the insoluble support and the polymerizable monomer to advance the polymerization reaction. Is done. The polymerization reaction is not limited, but for example, an insoluble support is put into a solvent in which a polymerizable monomer and a polymerization initiator are dissolved, and is stirred for 1 hour at a temperature of 0 ° C. or higher and 80 ° C. or lower. The heating is performed for 30 hours or less. Thereafter, the insoluble support is filtered under reduced pressure, washed and dried.

The use ratio of the insoluble support, the polymerizable monomer, and the polymerization initiator is not particularly limited, but is usually 0.1 mmol or more and 10 mmol of the polymerizable monomer with respect to 1 g of the insoluble support.
Hereinafter, the polymerization initiator is used at a ratio of 0.01 mmol to 10 mmol.

  Any solvent may be used as long as each polymerizable monomer can be dissolved, for example, alcohols such as methanol, ethanol, isopropanol, n-butanol, t-butyl alcohol, and n-pentanol, benzene, toluene, and tetrahydrofuran. , Dioxane, dichloromethane, chloroform, cyclohexanone, N, N-dimethylformamide, dimethyl sulfoxide, methyl acetate, ethyl acetate, butyl acetate, methyl ethyl ketone, methyl butyl ketone, ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, etc. Can be mentioned. These solvents are used alone or in combination of two or more.

  The polymerization initiator is not particularly limited, and examples thereof include 2,2′-azobisisobutylnitrile (hereinafter sometimes abbreviated as “AIBN”), 1,1′-azobis (cyclohexane-1-carbonitrile), and the like. Examples thereof include azo compounds, benzoyl peroxide, lauryl peroxide, organic peroxides such as tert-butyl peroxide, redox initiators such as hydrogen peroxide and ferrous ions.

  On the other hand, when immobilizing a polymer polymerized in advance on the surface of the insoluble support, a method of physically adsorbing the polymer polymerized in advance on the insoluble support or a method of chemical bonding can be mentioned. Preferably, in the polymer, a component that easily adsorbs to the insoluble support or a component having a functional group capable of reacting with a reactive functional group present on the surface of the insoluble support is incorporated as a copolymer during the polymerization of the polymer. . For example, as a functional group capable of reacting with a reactive functional group present on the surface of an insoluble support, for example, a silanol group obtained by hydrolyzing a silane coupling agent is preferable because it has high reactivity. A covalent bond can be formed by dehydration condensation with a hydroxyl group, amino group, carbonyl group, silanol group or the like on the body surface. The polymerization reaction of the polymerizable monomer can be performed according to the above.

  By applying the polymer to the surface of the insoluble support, the polymer can be adsorbed or chemically bonded to the surface of the insoluble support. Examples of the application method include preparing a polymer solution, and known methods such as dipping and spraying. After coating, it is preferable to dry at room temperature or under heating. In the case of chemical bonding, it may be carried out under reaction conditions corresponding to each. Thereby, a polymer layer is formed on the surface of the insoluble support.

  FIG. 2 schematically shows an example of a method for synthesizing the polymer layer of the carrier according to the embodiment. The carrier in FIG. 2 is obtained by synthesizing a polymer layer having a betaine structure by introducing a chain transfer group such as a mercapto group into the surface of inorganic particles having a hydroxyl group on the surface of silica beads or the like as an insoluble support. First, a chain transfer group is introduced onto the surface of the inorganic particles using a silane coupling agent having a chain transfer group. At this time, a chain transfer group is introduced by dehydrating condensation of a silanol group generated by hydrolysis of a hydrolyzable group such as an alkoxy group of a silane coupling agent with a hydroxyl group on the surface of the inorganic particles to form a covalent bond. The Subsequently, the inorganic particles into which the chain transfer group is introduced and the (meth) acrylic monomer in which at least a part of the monomers have a betaine structure are radically polymerized by adding a polymerization initiator in an appropriate solvent. The chain transfer group introduced into the inorganic particles serves as a polymerization starting point, and the surface of the inorganic particles is covered with a polymer layer having a betaine structure to form a polymer layer.

(Use of purification agent)
1. Concentration of glycopeptide The purification agent according to the present embodiment can be suitably used for concentration of glycopeptide, and specifically captures the glycopeptide, so that it contains impurities such as free peptide fragments that do not have a sugar chain. Can be removed to concentrate the glycopeptide. Here, “concentration” means to increase the concentration or abundance ratio of glycopeptides compared to before concentration. For example, a sample in which a glycopeptide such as a glycoprotein degradation product and a contaminant such as a free peptide fragment are mixed. The selective recovery of glycopeptides, and the like.

  A glycopeptide is one of amino acids that constitute a peptide in a sugar chain in which one or more monosaccharides or derivatives thereof, or two or more monosaccharides and / or derivatives thereof are connected in a linear or branched form by glycosidic bonds. The purification agent according to this embodiment targets all glycopeptides for concentration. The sugar chains of glycopeptides are mainly composed of N-glycoside-linked sugar chains (N-type sugar chains) in which sugar chains are bonded to asparagine residues, and O-glycoside-linked sugar chains (O-type sugars) that bind to serine, threonine and the like. It is roughly divided into two types. The purification agent according to the present embodiment concentrates any glycopeptide, and the type, chain length, structure, etc. of the glycopeptide are not particularly limited. Therefore, the effects of contaminants such as free peptide fragments and peptides such as peptide regions of glycopeptides on glycopeptides with small molecular weight and O-linked sugar chains and glycopeptides with long peptide regions. The glycopeptide can be concentrated specifically and efficiently.

2. Concentration of sugar chain The purification agent according to the present embodiment can be suitably used for concentration of sugar chains, and in order to specifically capture sugar chains, impurities other than sugar chains (proteins, peptides, lipids, Salt, etc.) can be removed to concentrate the sugar chain. Here, “concentration” means to increase the concentration or abundance ratio of sugar chains as compared to before concentration. For example, from a sample in which sugar chains released from glycoprotein and contaminants such as peptide fragments are mixed, Selectively recovering and the like.

  The sugar chain is a compound in which one or more monosaccharides or derivatives thereof, or two or more monosaccharides and / or derivatives thereof are connected in a linear or branched form by glycosidic bonds, and the carrier according to the embodiment is Concentrate all sugar chains. Examples of monosaccharides constituting the sugar chain or derivatives thereof include glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, fucose, sialic acid, and arabinose, and their derivatives. It is done. Examples of sugar chains include monosaccharides and derivatives thereof, polysaccharides, glycoproteins, and sugar chains released or derived from complex carbohydrates such as glycopeptides, proteoglycans, and glycolipids. Not. In particular, the carrier according to the embodiment can efficiently concentrate small sugars such as monosaccharides and disaccharides. Here, the sugar chain constituting the glycoprotein is mainly an N-glycoside-linked sugar chain (N-type sugar chain) in which the sugar chain is bound to an asparagine residue, and an O-glycoside-linked sugar that is bound to serine, threonine, or the like. There are two types of chains (O-type sugar chains). The carrier according to the embodiment is intended to concentrate any sugar chain, and the type, chain length, structure, etc. of the sugar chain are not particularly limited. Therefore, O-linked sugar chains with a small molecular weight are minimized and the influence of contaminants (proteins, peptides, lipids, salts, etc.) other than sugar chains existing in a large amount is minimized, and sugar chains are specifically and efficiently It can be concentrated.

[Concentration method of glycopeptide]
In one embodiment, the present invention comprises contacting the purification agent described above with a glycopeptide having a sugar chain having a length longer than a monosaccharide and a sample containing an organic solvent, and having the purification agent longer than a monosaccharide. Adsorbing a glycopeptide having a sugar chain; and contacting the purification agent with water to elute a glycopeptide having a sugar chain of a length longer than that of a monosaccharide. A method for purifying a glycopeptide having a sugar chain is provided.

  The method for concentrating a glycopeptide according to the present embodiment includes a step of concentrating the glycopeptide using the purification agent according to the embodiment. Specifically, the glycopeptide is concentrated from a sample containing the glycopeptide. Since the hydrophilicity of the purification agent according to the embodiment is extremely improved, the glycopeptide has a higher hydrophilicity than the peptide. Has affinity for. On the other hand, since peptides have lower hydrophilicity than glycopeptides, their affinity for the purification agent according to the embodiment is low. Therefore, the purification agent according to the embodiment can capture and concentrate the glycopeptide specifically and efficiently. The glycopeptide concentration method according to the present embodiment targets any glycopeptide described above for concentration.

  The sample containing a glycopeptide is not particularly limited as long as it may contain a glycopeptide. For example, a sample obtained by subjecting a sample containing a glycoprotein to a glycoprotein fragmentation treatment is exemplified. be able to. Samples containing glycoproteins include biological samples and environmental samples, and examples include biological samples such as whole blood, serum, plasma, urine, saliva, feces, cerebrospinal fluid, cells and cell cultures, and tissues. . The biological sample may be an unpurified one, a glycoprotein purified by a known technique, or a product that has been subjected to treatments such as degreasing, desalting, and protein fractionation. Also good.

The glycoprotein fragmentation treatment is not particularly limited as long as it can fragment the protein portion of the glycoprotein, and can be performed by using a protease or the like. For example, trypsin, chymotrypsin, pepsin, V8 protease, pronase, proteinase K, lysyl endoprotease, promeline, thermolysin, ficin,
Examples include, but are not limited to, caspases and subtilisins.

  In order to facilitate cleavage by a protease, the glycoprotein in the sample may be denatured or reduced before the protease treatment. Examples of the modifier include surfactants and chaotropic agents, and examples of the reducing agent include β-mercaptoethanol, dithiothreitol, glutathione, tris-2-carboxyethylphosphine, and tributylphosphine. Although it is mentioned, it is not limited to these.

  In the glycopeptide concentration method according to the present embodiment, first, a sample containing the glycopeptide is brought into contact with the purification agent according to the embodiment, and the glycopeptide is captured by the carrier. Since the purification agent according to the embodiment has increased hydrophilicity, it can specifically capture glycopeptides by hydrophilic interaction, and peptide fragments and the like that are present in large amounts in a sample can be captured by the carrier. It remains in a free state.

  The retention of the glycopeptide by the purification agent according to the embodiment is proportional to the concentration of the organic solvent. Therefore, an organic solvent or a mixed solvent of an organic solvent and water can be used as a solvent for the reaction solution when the purifying agent captures the glycopeptide. The solvent can be appropriately selected depending on the type of glycopeptide to be concentrated. The organic solvent is not particularly limited as long as it can dissolve the glycopeptide. For example, acetonitrile, tetrahydrofuran, acetone, dioxane, pyridine, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, etc. Can be mentioned. Preferably, organic solvents such as 1-butanol and ethanol are suitably used. Various buffers can be used for pH adjustment. Moreover, when using the mixed solvent of an organic solvent and water, the mixing ratio of an organic solvent and water is 3: 1-10: 1 by volume ratio, for example.

  Subsequently, the purification agent that has captured the glycopeptide is washed as necessary. The purification agent is preferably fixed to an insoluble support. By washing, substances other than the glycopeptide trapped on the carrier, particularly free peptide fragments can be removed. For example, the above-described solvent can be used as the cleaning liquid.

  After washing, the glycopeptide can be specifically concentrated by releasing the glycopeptide from the carrier that has captured the glycopeptide.

  The eluent for releasing the glycopeptide can use an organic solvent or a mixed solvent of an organic solvent and water, and can be appropriately selected depending on the type of glycopeptide or polymer to be concentrated. As the organic solvent, those described above can be used. In this step, the glycopeptide can be efficiently released by using a solvent having increased hydrophilicity. For example, it is possible to use only water without using an organic solvent, or it is possible to use a mixed solvent of an organic solvent and water. When using a mixed solvent, it is mentioned that the organic solvent is 3 times or less in volume ratio with respect to water.

  The glycopeptide concentration method according to this embodiment can be carried out by selecting a known concentration form using a carrier. Examples thereof include a batch method and a spin column method, but are not limited thereto. Although the batch method and the spin column method will be described in detail, the reagents and reaction conditions are as described above.

(Batch method)
In the case of concentration by the batch method, the sample containing the glycopeptide and the purification agent according to the embodiment are immersed in a reaction solution in an appropriate container (for example, a microtube, a centrifuge tube, a microplate, etc.) Capture the glycopeptide. The purification agent is preferably fixed to an insoluble support. Subsequently, the carrier-glycopeptide complex is subjected to solid-liquid separation, and the liquid phase portion containing impurities such as free peptide fragments is removed to recover the carrier. Solid-liquid separation can be performed by gravity sedimentation, centrifugation, or the like, and the liquid phase portion can be removed by suction or the like. In addition, solid-liquid separation may be performed by accumulating the carrier using magnetic force by including a magnetic material such as ferrite in the insoluble support of the carrier. In that case, it is not necessary to perform centrifugation or the like. In addition, solid-liquid separation may be performed by filtration through a filter, and in that case, it may be performed under reduced pressure or under pressure.

  Subsequently, the carrier capturing the glycopeptide is washed. By washing, impurities other than the glycopeptide trapped on the carrier, particularly free peptide fragments can be removed. The washing can be carried out by immersing the carrier capturing the glycopeptide in the washing solution in the above-mentioned appropriate container and repeating the exchange of the washing solution. For example, washing can be carried out by placing the carrier capturing the glycopeptide in a suitable container, adding a washing solution, shaking or stirring, and then repeating the operation of removing the liquid phase portion by solid-liquid separation. Solid-liquid separation can be performed as described above.

  After washing, the glycopeptide is released from the carrier that has captured the glycopeptide. The glycopeptide can be released by immersing the carrier in an eluate. For example, after sufficiently removing the washing solution, an appropriate amount of the eluate is added to the carrier on which the glycopeptide has been captured and shaken or stirred. Subsequently, the carrier is recovered by solid-liquid separation, and the eluate is collected in a new suitable container (for example, a collection tube or a collection plate). Solid-liquid separation can be performed as described above. If necessary, the glycopeptide can be concentrated by distilling off the eluate.

(Spin column method)
In the case of concentration by the spin column method, it can be carried out using a container having a filter or the like, for example, a filter cup. As the filter cup, for example, a filter cup having openings at the top and bottom and the bottom opening covered with a filter can be used. When using a filter cup, a sample solution containing a glycopeptide is put into a filter cup filled with the purification agent according to the embodiment, and the carrier and the sample are brought into contact with each other under a reaction solution by passing the solution. The purification agent is preferably fixed to an insoluble support. The liquid may be naturally dropped by gravity, centrifuged, or the like, or may be allowed to flow under reduced pressure or under pressure. After passing through, the drainage liquid containing free peptide fragments that have passed through the carrier is removed.

  Subsequently, the carrier capturing the glycopeptide is washed. By washing, impurities other than the glycopeptide captured on the carrier, particularly free peptide fragments can be removed. Washing can be performed by passing a washing solution through the carrier in the filter cup, and washing can be performed continuously from the glycopeptide capture reaction. The liquid flow can be performed as described above.

  After washing, the glycopeptide is released from the carrier that has captured the glycopeptide. The glycopeptide can be released by passing the eluate through the carrier in the filter cup, and can be continuously performed from the glycopeptide capture reaction through a washing operation. The eluate after passing through the carrier is collected in an appropriate container (for example, a collection tube or a collection plate). The liquid flow can be performed as described above. If necessary, the glycopeptide can be concentrated by distilling off the eluate.

  The glycopeptide concentrated by the glycopeptide concentration method according to this embodiment can be used as it is for analysis means such as glycopeptide structural analysis.

[Method for concentrating sugar chains]
In one embodiment, the present invention comprises contacting the purification agent described above with a sample containing a sugar chain having a length longer than a monosaccharide and an organic solvent, and a sugar chain having a length longer than a monosaccharide to the purification agent. A method for purifying a sugar chain having a length longer than that of a monosaccharide is provided, which comprises adsorbing a saccharide and elution of a sugar chain having a length longer than that of a monosaccharide by contacting the purification agent with water.

  The sugar chain concentration method according to this embodiment includes a step of concentrating sugar chains using the purification agent according to this embodiment. Specifically, sugar chains are concentrated from a sample containing sugar chains, and the purification agent according to the present embodiment has a very improved hydrophilicity, and therefore has a higher hydrophilicity than peptides and lipids. Has affinity for high sugar chains. On the other hand, since peptides and lipids have a lower hydrophilicity than sugar chains, the affinity for the purification agent according to this embodiment is low. Therefore, the purification agent according to the present embodiment can specifically and efficiently capture and concentrate sugar chains. The sugar chain concentration method according to the present embodiment targets any sugar chain as described above.

  The sample containing sugar chains is not particularly limited as long as it is a sample that may contain sugar chains. For example, in addition to samples containing sugar chains themselves such as monosaccharides and polysaccharides, And those obtained by subjecting a sample containing glycoconjugates such as glycoproteins to sugar chain release treatment. Samples containing complex sugar chains such as monosaccharides, polysaccharides and glycoproteins include biological samples and environmental samples, such as whole blood, serum, plasma, urine, saliva, stool, cerebrospinal fluid, cells and cells. Examples include biological samples such as cultures and tissues. The biological sample may be unpurified, or may be a product obtained by purifying complex sugars such as monosaccharides, polysaccharides and glycoproteins by a known technique, or degreasing, desalting, and protein fractionation. Alternatively, it may be subjected to a treatment such as heat denaturation.

  The sugar chain releasing treatment from the complex saccharide such as glycoprotein is not particularly limited as long as it can release the sugar chain from the complex sugar, and there is no limitation on enzymatic treatment and chemical treatment. For example, peptide N-glycanase (PNGase F, PNGase A) digestion, endo-β-N acetylglucosaminidase (Endo-H, Endo-F, Endo-A, Endo-M) digestion, endo O-glycanase digestion, hydrazine degradation, Although it can be carried out by trifluoroacetic acid decomposition, β elimination by alkali treatment or the like, it is not limited thereto. Prior to the sugar chain releasing treatment, the protein and peptide portion may be fragmented with the above-described protease or the like on the glycoprotein or glycopeptide.

  In the sugar chain concentration method according to this embodiment, first, a sample containing a sugar chain is brought into contact with the purification agent according to the embodiment, and the purification agent captures the sugar chain. The purification agent is preferably fixed to an insoluble support. Since the carrier according to the present embodiment has a high degree of hydrophilicity, it can specifically capture sugar chains by hydrophilic interaction, and impurities (proteins) other than sugar chains present in a large amount in the sample. , Peptides, lipids, salts, etc.) remain in a free state without being captured by the carrier.

  The sugar chain retention by the carrier according to this embodiment is proportional to the organic solvent concentration. Therefore, an organic solvent or a mixed solvent of an organic solvent and water can be used as a solvent for a reaction solution when the carrier captures a sugar chain. The solvent can be appropriately selected depending on the type of sugar chain to be concentrated. The organic solvent is not particularly limited as long as it can dissolve the sugar chain, and examples thereof include acetonitrile, tetrahydrofuran, acetone, dioxane, pyridine, methanol, ethanol, 1-propanol, 2-propanol, and 1-butanol. Can be mentioned. Preferably, an organic solvent such as acetonitrile, 1-butanol or ethanol is suitably used. Various buffers can be used for pH adjustment. When a mixed solvent of an organic solvent and water is used, the mixing ratio of the organic solvent and water is, for example, 50:50 to 100: 0 by volume, preferably 90:10 to 100: 0, more preferably 95: 5 to 100: 0.

  Subsequently, the carrier capturing the sugar chain is washed as necessary. By washing, contaminants (protein, peptide, lipid, salt, etc.) other than the sugar chain captured by the carrier can be removed. For example, the above-described solvent can be used as the cleaning liquid.

  After washing, the sugar chain can be specifically concentrated by re-releasing the sugar chain from the carrier that has captured the sugar chain.

  As an eluent for re-releasing sugar chains, an organic solvent or a mixed solvent of an organic solvent and water can be used, and can be appropriately selected depending on the type of sugar chain or polymer to be concentrated. As the organic solvent, those described above can be used. In this step, sugar chains can be efficiently released by using a solvent having increased hydrophilicity. For example, it is possible to use only water without using an organic solvent, or it is possible to use a mixed solvent of an organic solvent and water. In the case of using a mixed solvent, the organic solvent is 3 times or less in volume ratio with respect to water, but it is particularly preferable to use only water without using the organic solvent.

  The sugar chain concentration method according to this embodiment can be carried out by selecting a known concentration form using a carrier. Examples thereof include a batch method and a spin column method, but are not limited thereto. The procedure of the batch method and the spin column method can be performed in accordance with the procedure of the above [Method of concentrating glycopeptide], and the reagents and reaction conditions are as described above.

  The sugar chain concentrated by the sugar chain concentration method according to this embodiment can be used as it is for analysis means such as sugar chain structure analysis, and can be labeled as necessary.

[Glycopeptide enrichment kit]
The kit for concentrating glycopeptides according to the present embodiment includes the purification agent according to the embodiments, a protocol for using the kit, and the like as instructions for use. The purification agent is preferably fixed to an insoluble support. The instruction manual may be written or printed on paper or other media, or may be recorded on an electronic medium such as a magnetic tape, a magnetic disk, or an optical disk. In this way, reagents necessary for carrying out the glycopeptide concentration method can be provided as a kit.

  The kit for concentrating glycopeptides according to this embodiment can further contain reagents and containers necessary for carrying out the kit. For example, a reaction solution for capturing a glycopeptide by a carrier according to the present embodiment, a buffer solution for adjusting pH and the like during the reaction, a washing solution for washing, and releasing the glycopeptide from the carrier that has captured the glycopeptide For example. These may be provided in the form of lyophilized powder, in which case the kit for concentrating glycopeptides may further contain a diluent for dilution in use. Moreover, containers, such as a filter cup, a multiwell plate, a filter plate, and a microtube, may be included, and the carrier according to the present embodiment may be included in a state of being filled in these containers. The definition of each term and preferred embodiments are as described above. In this way, the glycopeptide can be concentrated more easily by using a kit containing carriers, reagents and information necessary for the concentration of the glycopeptide.

[Kit for sugar chain concentration]
The kit for sugar chain concentration according to the present embodiment includes the purification agent according to the embodiment, a protocol for using the kit, and the like as instructions for use. The purification agent is preferably fixed to an insoluble support. The instruction manual may be written or printed on paper or other media, or may be recorded on an electronic medium such as a magnetic tape, a magnetic disk, or an optical disk. Thus, the reagents necessary for carrying out the sugar chain concentration method can be provided as a kit.

  The kit for sugar chain concentration according to this embodiment can further include reagents and containers necessary for carrying out the kit. For example, a reaction solution for capturing a sugar chain by the carrier according to the present embodiment, a buffer solution for adjusting pH and the like during the reaction, a washing solution for washing, and re-releasing the sugar chain from the carrier that has captured the sugar chain For example, an eluate for the treatment. These may be provided in the form of lyophilized powder. In that case, the kit for sugar chain concentration may further contain a diluent for dilution in use. Moreover, containers, such as a filter cup, a multiwell plate, a filter plate, and a microtube, may be included, and the carrier according to the present embodiment may be included in a state of being filled in these containers. The definition of each term and preferred embodiments are as described above. In this way, the sugar chain can be more easily concentrated by preparing a carrier, reagents, information and the like necessary for the sugar chain concentration.

〔apparatus〕
In one embodiment, the present invention provides a sugar chain or a monosaccharide having a length longer than that of a monosaccharide, comprising a container holding unit that holds a container containing the above-described purification agent, and a reagent introduction unit that introduces a reagent into the container. An apparatus for purifying a glycopeptide having a sugar chain having the above length is provided. The purification agent is preferably fixed to an insoluble support.

  FIG. 9 is a schematic diagram for explaining the apparatus of the present embodiment. Note that the configuration of the apparatus described below is merely an example, and the apparatus of the present embodiment is not limited to this configuration. As shown in FIG. 9, the apparatus 100 includes a container holding unit 20 that holds a container 15 that contains a carrier 10, and a reagent introduction unit 30 that introduces a reagent into the container 15. In the example of FIG. 9, the container 15 is attached to a collection container 16 (for example, a collection tube, a collection plate, etc.) described later.

  The reagent introduction unit 30 includes a sample introduction unit 35 that introduces a sample 31 containing a sugar peptide having a sugar chain longer than a monosaccharide or a sugar peptide having a sugar chain longer than a monosaccharide and an organic solvent into the container 15, and the container 15. The cleaning liquid introducing part 35 for introducing the cleaning liquid 32 into the container 15 and the eluent introducing part 35 for introducing the eluent 33 into the container 15 are included. In this example, the sample introduction part, the cleaning liquid introduction part, and the eluate introduction part are composed of the same member 35.

  The container holding part 20 is for holding the container 15 containing the carrier 10. The container holding unit 20 may directly hold the container 15 or may hold another member (collection container 16) that holds the container 15 as shown in FIG. The aspect in which the container holding part 20 holds the container 15 is not limited to this, and for example, an aspect in which most of the container is fitted and held in the holding hole or the holding hole of the container holding part 20 may be mentioned. In addition to this, the container holding portion is held by the holding portion of the container holding portion. And the like.

  The reagent introduction unit 30 is for introducing liquids into the container 15 held by the container holding unit 20. In the example of FIG. 9, the reagent introduction unit 30 includes a tank 34 that contains a sample 31, a cleaning liquid 32, and an eluate 33, a liquid feeding pipe 35 a that feeds each reagent contained in the tank 34, and a liquid feeding of each reagent. And the introduction part 35 for introducing each reagent into the container 15.

  The sample introduction unit 35, the cleaning solution introduction unit 35, and the eluate introduction unit 35 add the sample 31, the cleaning solution 32, and the elution solution 33 in the same reaction vessel 15. The aspect in which the reagent introduction part 30 introduces the liquid into the reaction container 15 is not particularly limited. For example, the reaction is performed from the liquid supply source (31, 32, 33) in which the liquid to be supplied is stored via the tubular member. An embodiment in which the liquid is fed into the container 15 is exemplified. In addition to this, a mode in which the liquid collected in the tubular member is injected into the reaction container, and the like can be mentioned.

  The apparatus 100 may further include a solid-liquid separation unit 40 that solid-liquid separates the contents of the container 15. When the apparatus 100 includes the solid-liquid separation unit 40, the solid-liquid separation unit 40 separates the solid and the liquid from the contents contained in the container 15. The solid is left in the container 15 and is substantially the carrier 10 and the substance fixed thereto. Further, the container 15 may be equipped with a collection container 16 (for example, a collection tube, a collection plate, etc.).

  The specific separation format of the solid-liquid separation unit 40 is not particularly limited, and examples thereof include centrifugation, reduced pressure, and pressurization. In the example of FIG. 9, the separation type of the solid-liquid separation unit 40 is centrifugal separation. The solid-liquid separation unit 40 includes a rack 41 that holds the container 15 (or 16), a drive shaft 42, and a motor 43.

  As in the example of FIG. 9, the solid-liquid separation unit 40 may be configured as a constituent member independent of the container holding unit 20. In this case, the apparatus 100 may include a container transfer unit 50 that automatically transfers the container 15 (and 16) from the container holding unit 20 to the solid-liquid separation unit 40. In the transfer of the containers 15 (and 16), the container transfer unit 50 may be configured such that only the container 15 is transferred, or the container 15 is transferred with the collection container 16 mounted. May be. The container transfer unit 50 includes an arm that operates to grip and open and move the container 15 directly or indirectly (that is, via the collection container 16), and an arm control unit that controls the operation of the arm. It may be comprised including.

  A glycopeptide having a sugar chain longer than a monosaccharide or a sugar chain longer than a monosaccharide fixed to the carrier 10 by activating the solid-liquid separation unit 40 by centrifugation, decompression, pressurization, or the like Can be left in the container 15 and the cleaning liquid can be discarded in the collection container 16. In the elution process, an eluate containing a glycopeptide having a sugar chain longer than a monosaccharide or a sugar chain longer than a monosaccharide can be recovered in the recovery container 16.

  The apparatus 100 may further include a temperature adjustment unit 60 that adjusts the temperature of the contents of the container 15. When the apparatus 100 includes the temperature adjustment unit 60, the temperature adjustment unit 60 may have at least a heater function.

  The apparatus 100 may be configured so that at least one of the operable components (for example, the liquid introduction unit 35, the arm 50, the solid-liquid separation unit 40, the temperature adjustment unit 60, and the liquid transfer unit 50), preferably all of them are automatically controlled. Good. As a result, a glycopeptide having a sugar chain longer than a monosaccharide or a sugar chain longer than a monosaccharide can be purified more rapidly.

  Hereinafter, the present invention will be described specifically by way of examples. However, the present invention is not limited to these examples.

<Synthesis example>
(Support in which a polymer containing a structural unit derived from 2-methacryloyloxyethyl phosphorylcholine is immobilized on silica beads)
In this synthesis example, the synthesis of a polymer containing a structural unit derived from 2-methacryloyloxyethyl phosphorylcholine (hereinafter referred to as “MPC polymer”) on the surface of silica beads will be described as an example. Is not intended to limit.

Introduction of chain transfer group into silica beads 5 g of (3-mercaptopropyl) trimethoxysilane (M0928, manufactured by Tokyo Chemical Industry Co., Ltd.), which is a silane coupling agent having a chain transfer group, is added to 50 mL of acetic acid aqueous solution at pH 3.0 and 50 mL of ethanol. And then hydrolyzing the silane coupling agent by stirring at room temperature for 1 hour, followed by silica beads as an example of an insoluble support (average particle size 5 μm, pore size 70 mm, Fuji After adding 5 g of SMB70-5 manufactured by Silysia Chemical Ltd. and stirring at 70 ° C. for 2 hours, silica beads were collected from the reaction solution by suction filtration and heated at 100 ° C. for 1 hour. Then, after dispersing with ethanol and shaking well, the supernatant was removed by centrifugation and dried.

Polymer Synthesis 2-Methacryloyloxyethyl phosphorylcholine (hereinafter referred to as “MPC monomer”, manufactured by Nippon Oil & Fats Co., Ltd.), which is a structural unit of the polymer, was dissolved in ethanol to prepare 20 mL of a 0.8 mol / L monomer solution. . AIBN was added there so that it might become 0.027 mol / L, and it stirred until it became uniform. Thereafter, 4 g of silica beads treated with the above methacryloxypropyldimethylmethoxysilane were added and reacted at 70 ° C. for 6 hours in an argon gas atmosphere. Next, the silica beads are collected from the reaction solution by centrifugation, dispersed in ethanol, shaken well, and then collected by suction filtration and dried to contain a structural unit derived from 2-methacryloyloxyethyl phosphorylcholine. A carrier in which the polymer was immobilized on silica beads (hereinafter simply referred to as “carrier”) was obtained.

(Measurement of the weight of the layer containing the polymer introduced on the surface of the carrier)
The weight of the polymer-containing layer introduced on the surface of the carrier obtained above was 10 ° C / 500 ° C from room temperature to 500 ° C in an air atmosphere using a TGA apparatus (TG / DTA6200 manufactured by Seiko Instruments Inc.). The temperature was reduced in minutes, and the weight loss rate after maintaining 500 ° C. for 1 hour was measured. From this value and the surface area of the particle per unit weight separately obtained by the BET method, the weight of the layer containing the polymer introduced on the surface of the particle was calculated to be 1.08 mg / m ² .

Example 1. Confirmation of physical properties (carrier)
In this example, the physical properties of the carrier prepared in the above <Synthesis Example> were confirmed from the viewpoint of solid-liquid separation physical properties.

  After adding 200 μL of ethanol: butanol (volume ratio 1: 5, referred to as solution A) mixed solution to 10 mg of the carrier prepared in the above <Synthesis example>, and vigorously dispersing by vortex, tabletop centrifuge (TOMY, PMC-060) And the degree of separation of the solid-liquid surface of the carrier was confirmed. It was confirmed that the carrier was sunk by centrifuging for a few seconds and the solid-liquid surface became clear. Since the carrier was white, it was confirmed that the solid-liquid surface was clear (Table 1).

Comparative Example 1 Confirmation of physical properties (Sepharose beads)
In this comparative example, the physical properties of Sepharose beads were confirmed from the viewpoint of solid-liquid separation physical properties, and compared with the physical properties of the carrier prepared in the above <Synthesis Example> by comparison with Example 1.

  200 μL of the solution A of Example 1 was added to 100 μL of Sepharose beads (SIGMA, Sepharose CL-4B), and the mixture was vigorously dispersed by vortexing, and then centrifuged with a tabletop centrifuge (TOMY, PMC-060), The degree of separation was confirmed. It was confirmed that the Sepharose beads were not yet settled by centrifugation for a few seconds. By continuing the centrifugation for 10 seconds or more, Sepharose beads were sunk, but it was confirmed that the solid-liquid interface was very unclear because the color was translucent (Table 1).

  From the results of Example 1 and Comparative Example 1, it was confirmed that the carrier prepared in the above <Synthesis Example> had excellent physical properties in solid-liquid separation, and could be easily, simply, and reliably separated into solid and liquid.

Example 2 Concentration of glycopeptide using carrier In this example, the concentration of glycopeptide from the sample was confirmed by both batch and spin columns using the carrier prepared in <Synthesis Example> above.

(1) Peptide fragmentation of protein 50 μL of ultrapure water, 5 μL of 1 M aqueous ammonium bicarbonate solution, and 5 μL of 120 mM dithiothreitol solution were added to 5 mg RNase B (SIGMA) and reacted at 60 ° C. for 30 minutes. Thereafter, 10 μL of 123 mM iodoacetamide aqueous solution was added, and the mixture was allowed to react at room temperature for 1 hour while being shielded from light. Trypsin 400 unit was added and allowed to react overnight. An appropriate amount of ultrapure water was added to prepare a protein concentration of 50 μg / μL.

(2) Recovery of glycopeptide In the case of a batch type, 1 mg of the beads was added to a 1.5 mL sample tube. After 200 μL of the solution A of Example 1 was added to disperse the beads, the solution was centrifuged for several seconds using a tabletop centrifuge. After removing the solution, add the sample solution (1 μL of the solution prepared in (1) above, 99 μL of ultrapure water, 600 μL of the solution A of Example 1) to the tube containing the carrier prepared in the above <Synthesis Example>. Stir vigorously. Then, it centrifuged using the tabletop centrifuge, the support | carrier was settled, and the solution was removed. Subsequently, 700 μL of ethanol: butanol: water (volume ratio 1: 5: 1, used as a washing solution) was added and stirred vigorously, and then the carrier was precipitated by centrifugation to remove the solution. Furthermore, 700 μL of 50% ethanol aqueous solution was added and stirred vigorously, and then the carrier was precipitated by centrifugation, and the solution was recovered. The collected solution was dried using a centrifugal evaporator and redissolved in 13 μL of ultrapure water.

B. In the case of a spin column: 1 mg of the carrier prepared in the above <Synthesis Example> was added to a spin column (Ultrafree-MC, Millipore Cat #: UFC30HVNB). After adding 200 μL of the solution A of Example 1, the solution was removed by centrifugation for several seconds using a tabletop centrifuge. Add the sample solution (1 μL of the solution prepared in (1) above, 99 μL of ultrapure water, 600 μL of the solution A of Example 1) to the spin column containing the carrier, and centrifuge using a tabletop centrifuge to remove the solution. did. Subsequently, 700 μL of washing solution was added, and the solution was removed by centrifugation. Further, 700 μL of 50% ethanol aqueous solution was added, and the solution was collected by centrifugation. The collected solution was dried using a centrifugal evaporator and redissolved in 13 μL of ultrapure water.

(3) LC-MS analysis LC-MS analysis was performed using the conditions shown in Table 2 using 3 μL of each solution prepared in (2) above. The obtained total ion chromatogram is shown in FIG. In FIG. 3, the horizontal axis represents the retention time (minutes), and the vertical axis represents the signal intensity (relative value). It was confirmed that many signals were present in the sample before the glycopeptide recovery process (upper figure in FIG. 3). On the other hand, in the sample solution from which the glycopeptide was recovered by the method of the present invention, it was confirmed that the signal was significantly reduced and the main signal was unified (lower figure in FIG. 3). The MS results of this main peak (elution time 5.2 to 5.8 minutes) are shown in FIG. In FIG. 4, the horizontal axis represents the m / z value, and the vertical axis represents the signal intensity (relative value). A number of equidistant peaks corresponding to one hexose were detected, and it was confirmed that the peak was derived from a peptide bound with high mannose, which is a typical sugar chain of RNase B. It was confirmed that glycopeptides can be easily concentrated by using the method of the present invention.

Example 3 Concentration of sugar chains using a carrier In this example, the concentration of sugar chains from a sample was confirmed using a spin column using the carrier prepared in <Synthesis Example> above.

(1) Preparation of sugar chain solution Glucose oligomer (manufactured by Seikagaku Corporation, product number: 800111) prepared from a hydrolyzate of dextran was dissolved in ultrapure water to a concentration of 1 mg / mL to obtain a sugar chain solution. .

(2) Recovery of sugar chain 10 mg of the carrier prepared in the above <Synthesis Example> was added to a spin column (Ultrafree-MC, Millipore Cat #: UFC30HVNB). After adding 200 μL of ultrapure water, the solution was removed by centrifugation for several seconds using a tabletop centrifuge. After adding 990 μL of acetonitrile to 10 μL of the solution prepared in the above (1) and mixing well, it was added to a spin column containing a carrier and centrifuged using a tabletop centrifuge to remove the solution. Subsequently, 600 μL of acetonitrile was added, and the solution was removed by centrifugation. Again, 600 μL of acetonitrile was added, and the solution was removed by centrifugation. After adding 100 μL of ultrapure water, the solution was collected by centrifugation. The collected solution was dried using a centrifugal evaporator.

(3) Labeling of the recovered sugar chain The dried product obtained in (2) above is added to a 2AB labeling solution (0.35M 2-aminobenzamide, 30% acetic acid / 70% dimethylsulfone so that it becomes 1M sodium cyanoborohydride. 10 μL of a solution dissolved in a xoxide solvent) was added. After reacting at 60 degrees for 2 hours, 90 μL of ultrapure water was added.

(4) HPLC analysis Using 1 μL of each solution prepared in (3) above, HPLC analysis was performed using the conditions shown in Table 2. Among the obtained peaks, the area values of the peaks corresponding to glucose monosaccharide to sugar 10 were confirmed. The obtained results are shown in FIG. The control sample refers to a sample obtained by drying only 10 μL of the glucose oligomer solution and then performing only the step (3). The results were almost the same between the sample subjected to sugar chain recovery using the carrier and the control sample, and it was confirmed that there was almost no loss of sugar chain when the sugar chain was recovered using the carrier.

Comparative Example 2 Concentration of sugar chains using a cleanup column In this comparative example, sugar chains were concentrated using a conventionally known cleanup column (a column containing a silica-based carrier). Comparison with sugar chain concentration using the prepared carrier was performed.

(1) Preparation of sugar chain solution The procedure was the same as in Example 3.

(2) Recovery of sugar chains After adding 200 μL of ultrapure water to a cleanup column (Sumitomo Bakelite, BS-45403 accessory), the solution was removed by centrifugation for several seconds using a tabletop centrifuge. After adding 990 μL of acetonitrile to 10 μL of the solution prepared in the preparation of the sugar chain solution (above (1)) and mixing well, it was added to the cleanup column and centrifuged using a tabletop centrifuge to remove the solution. Subsequently, 600 μL of acetonitrile was added, and the solution was removed by centrifugation. Again, 600 μL of acetonitrile was added, and the solution was removed by centrifugation. After adding 100 μL of ultrapure water, the solution was collected by centrifugation. The collected solution was dried using a centrifugal evaporator.

(3) Labeling of the recovered sugar chain The procedure was the same as in Example 3.

(4) HPLC analysis It carried out by the same procedure as Example 3, and confirmed the area value of the peak corresponding to glucose 1 sugar-10 sugar among the obtained peaks. The obtained result is shown in FIG. The control sample refers to a sample obtained by drying only 10 μL of the glucose oligomer solution and then performing only the step (3). Data showing that the loss of monosaccharides and disaccharides was large from the samples subjected to sugar chain recovery using the cleanup column. It was confirmed that a conventionally known cleanup column is insufficient for recovering small sugar chains such as O-type sugar chains.

Comparative Example 3 Concentration of sugar chains using graphite carbon In this comparative example, sugar chains were concentrated using a conventionally known graphite carbon, and the sugar chains were concentrated using the carrier prepared in the above <Synthesis example> in Example 3. A comparison was made.

(1) Preparation of sugar chain solution The procedure was the same as in Example 3.

(2) Recovery of sugar chain 1 mL of acetonitrile was passed through a graphite carbon column (Sigma Aldrich, Superclin ENVI-Carb C). Furthermore, 3 mL of water was passed through. Subsequently, 10 μL of the solution prepared in the preparation of the sugar chain solution (above (1)) and 90 μL of 0.1% acetic acid water were mixed and passed through a graphite carbon column. 3 mL of water was passed through to wash the graphite carbon. After mounting a 0.22 μm filter, 100 μL of ultrapure water was passed through to collect the solution. The collected solution was dried using a centrifugal evaporator.

(3) Labeling of the recovered sugar chain The procedure was the same as in Example 3.

(4) HPLC analysis It carried out by the same procedure as Example 3, and confirmed the area value of the peak corresponding to glucose 1 sugar-10 sugar among the obtained peaks. The obtained results are shown in FIG. The control sample refers to a sample obtained by drying only 10 μL of the glucose oligomer solution and then performing only the step (3). In the sample where glycan recovery was performed using the graphite carbon column, sugar loss occurred overall, and data indicating that no saccharide could be recovered from the monosaccharide or disaccharide was obtained from the sample. It was. It has been confirmed that conventionally known graphite carbon is not particularly suitable for recovering small sugar chains (monosaccharide, disaccharide, etc.).

  As a result of comparison with the results of Example 3 and the results of Comparative Examples 2 and 3, the carrier prepared in the above <Synthesis Example> has almost no loss when recovering the sugar chain, which is difficult with the prior art. It can be understood that the present invention can also be suitably used to recover small sugar chains (monosaccharides, disaccharides, etc.).

Example 4 Concentration of sugar chains using a purification agent In this example, a purification agent in which a betaine structure represented by the following formula (2) was introduced into a crosslinked polystyrene skeleton was used to concentrate sugar chains from a sample using a spin column. confirmed.

(1) Preparation of sugar chain solution The procedure was the same as in Example 3.

(2) Recovery of sugar chain 10 mg of the above-mentioned purification agent was added to a spin column (Ultrafree-MC, Millipore Cat #: UFC30HVNB). After adding 200 μL of acetonitrile, the solution was removed by centrifugation for several seconds using a tabletop centrifuge. After adding 990 μL of acetonitrile to 10 μL of the solution prepared in the above (1) and mixing well, it was added to a spin column containing a purifying agent and centrifuged using a tabletop centrifuge to remove the solution. Subsequently, 600 μL of acetonitrile was added, and the solution was removed by centrifugation. Again, 600 μL of acetonitrile was added, and the solution was removed by centrifugation. After adding 100 μL of ultrapure water, the solution was collected by centrifugation. The collected solution was dried using a centrifugal evaporator.

(3) Labeling of the recovered sugar chain The procedure was the same as in Example 3.

(4) HPLC analysis It carried out by the same procedure as Example 3, and confirmed the area value of the peak corresponding to glucose 1 sugar-10 sugar among the obtained peaks. The obtained result is shown in FIG. The control sample refers to a sample obtained by drying only 10 μL of the glucose oligomer solution and then performing only the step (3). It was confirmed that the sample subjected to sugar chain recovery with the purifying agent was able to recover small sugars such as monosaccharides and disaccharides although there was some loss compared to the control sample.

  The present invention can provide a technique capable of specifically and efficiently capturing hydrophilic glycopeptides and sugar chains according to the present invention. Therefore, the present invention is a technical field that requires concentration of glycopeptides and sugar chains, for example, the onset mechanism of various diseases accompanied by sugar chain structural changes, development of disease treatment and diagnostic techniques, It can be used in technical fields such as drug discovery.

  DESCRIPTION OF SYMBOLS 100 ... The apparatus which prepares the sugar chain of glycoprotein, 10 ... Carrier, 15 ... Container, 16 ... Recovery container, 20 ... Container holding part, 30 ... Reagent introduction part, 31 ... Sample, 32 ... Washing liquid, 33 ... Elution liquid, 34 ... Tank, 35 ... Sample introduction part (cleaning liquid introduction part, eluate introduction part), 35a ... Liquid feed pipe, 36, 37, 38 ... Valve, 40 ... Solid-liquid separation part, 41 ... Rack, 42 ... Drive shaft, 43 ... motor, 50 ... container transfer part (liquid transfer part), 60 ... temperature control part.

Claims (16)

  A purification agent for a glycopeptide having a sugar chain longer than a monosaccharide or a sugar chain longer than a monosaccharide, comprising a compound having a betaine structure. The purification agent according to claim 1, wherein the betaine structure is a structure represented by the following formula (1).

[In the formula (1), Z represents a cationic group selected from the group consisting of a secondary amino group, a tertiary amino group, a quaternary ammonium group and an imino group, and L represents an alkylene group having 1 to 10 carbon atoms. A represents an anionic group selected from the group consisting of a phosphate group, a carboxyl group, a phosphonic acid group, a phosphinic acid group, a sulfonic acid group, a sulfinic group, a sulfenic group, a hydroxyl group, a thiol group and a boronic acid group. Represent. ]   The purification agent according to claim 1 or 2, wherein the cationic group is a quaternary ammonium group.   The purification agent according to any one of claims 1 to 3, wherein the anionic group is a phosphate group.   The purification agent according to any one of claims 1 to 4, wherein the betaine structure is a phosphorylcholine group.   The purification agent according to any one of claims 1 to 5, wherein the compound is a polymer in which a side chain having a betaine structure is bonded to a main chain.   The purification agent according to claim 6, wherein the polymer is a polymer of a monomer containing a (meth) acrylic compound.   Purification of a glycopeptide having a sugar chain having a length longer than a monosaccharide or a sugar chain having a length longer than a monosaccharide, wherein the purification agent according to any one of claims 1 to 5 is fixed to an insoluble support. Carrier.   A carrier for purifying a glycopeptide having a sugar chain longer than a monosaccharide or a sugar chain longer than a monosaccharide, wherein the purification agent according to claim 6 or 7 is fixed to an insoluble support. The carrier according to claim 9, wherein the weight of the polymer fixed to the insoluble support is 0.5 mg to 1.5 mg per unit surface area (m ² ) of the insoluble support.   The purification carrier according to any one of claims 8 to 10, wherein the insoluble support is composed of an inorganic substance.   The support | carrier for refinement | purification as described in any one of Claims 8-11 whose specific gravity is 1.05-3.00.   The support | carrier for refinement | purification as described in any one of Claims 8-12 whose shape is spherical and whose average particle diameter is 0.5 micrometer-100 micrometers. The purification agent according to any one of claims 1 to 7, or the purification carrier according to any one of claims 8 to 13, a sugar chain having a length longer than a monosaccharide, or a length longer than a monosaccharide. Contacting a glycopeptide having a sugar chain with a sample containing an organic solvent, and adsorbing the sugar chain or the glycopeptide to the purification agent or purification carrier;
Contacting the purification agent or purification carrier with water and eluting a glycopeptide having a sugar chain longer than a monosaccharide or a sugar chain longer than a monosaccharide.
A method for purifying a glycopeptide having a sugar chain longer than a monosaccharide or a sugar chain longer than a monosaccharide.   A purification agent according to any one of claims 1 to 7 or a carrier for purification according to any one of claims 8 to 13 and a length longer than a monosaccharide comprising protocol information for use of the kit. A kit for purifying a glycopeptide having a sugar chain or a sugar chain longer than a monosaccharide. A container holding unit for holding a container containing the purification agent according to any one of claims 1 to 7 or the purification carrier according to any one of claims 8 to 13;
A reagent introduction part for introducing reagents into the container;
An apparatus for purifying a glycopeptide having a sugar chain longer than a monosaccharide or a sugar chain longer than a monosaccharide.

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